Biomechanical investigation of flexor digitorum tendons in trigger finger patients using sonography

Trigger finger (TF) has generally been ascribed to primary changes in the first annular (A1) pulley. Repeated friction between the A1 pulley and flexor digitorum tendons could result in swelling of soft tissues, and thus it has been speculated that TF affects tendons’ biomechanical behaviors. However, the pathology mechanism related to these behaviors remains unclear. The purposes of this study are to understand (1) the variations in the morphologies of the flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS) between normal fingers and TFs, (2) the differences in the biomechanical behaviors of the FDP and FDS between normal fingers and TFs in various finger flexion positions, and (3) the effect of various finger positions on the biomechanical behaviors of the FDP and FDS

Host non-inflammatory neutrophils mediate the engraftment of bioengineered vascular networks

Notwithstanding remarkable progress in vascular network engineering, implanted bioengineered microvessels largely fail to form anastomoses with the host vasculature. Here, we demonstrate that implants containing assembled human vascular networks (A-Grafts) fail to engraft due to their inability to engage non-inflammatory host neutrophils upon implantation into mice. In contrast, unassembled vascular cells (U-Grafts) readily engage alternatively polarized neutrophils, which in turn serve as indispensable mediators of vascular assembly and anastomosis. The depletion of host neutrophils abrogated vascularization in U-Grafts, whereas an adoptive transfer of neutrophils fully restored vascularization in myeloid-depleted mice. Neutrophil engagement was regulated by secreted factors and was progressively silenced as the vasculature matured. Exogenous addition of factors from U-Grafts reengaged neutrophils and enhanced revascularization in A-Grafts, a process that was recapitulated by blocking Notch signaling. Our data suggest that the pro-vascularization potential of neutrophils can be harnessed to improve the engraftment of bioengineered tissues

Functional studies of dendritic cells in type 1 diabetes: non-obese diabetic (NOD) mouse model

© 2012 Dr. Chin Nien LeeDendritic cells (DC) are highly efficient antigen presenting cells and are important in regulating immune defense and tolerance. Both conventional DC (cDC) and plasmacytoid DC (pDC) are found in lymphoid tissues, such as the thymus and spleen. cDC can be further segregated into two populations: the CD8α+Sirpα- (CD8+) and CD8α-Sirpα+ (CD8-) cDC. Thymic cDC play an important role in the negative selection of auto-reactive T cells and the induction of naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTregs). The splenic cDC, particularly the CD8+ cDC, can cross-present auto-antigen in steady state and delete auto-reactive T cells in peripheral tissues. In addition, pDC have also been shown to induce or regulate immune tolerance in various autoimmune disease models via cross talk with regulatory T cells, producing anti-inflammatory cytokines, including TGF-β, IL-10 or typtophan-catabolising enzyme indoleamine 2,3-dioxygenase (IDO) which can inhibit the activity of T cells. Human type 1 diabetes (T1D) mellitus is an autoimmune disease that results from the autoimmune destruction of β-cells in the pancreas. The non-obese diabetes (NOD) mouse is an important animal model for T1D and shares many pathological features with human T1D. Both genetic and environmental factors are believed to play an important role in T1D development. However, the mechanisms for disease initiatory and the loss of immune tolerance remain unclear. In addition, infectious pathogens such as Lymphocytic Choriomeningitis virus (LCMV), mycobacterial reagents (e.g. BCG, CFA) or CpG oligonucleotide can delay or prevent the onset of diabetes in NOD mice. Again, the mechanisms for these effects remain elusive. Until recently, no thorough studies have been performed on the phenotype and function of steady state DC subsets in autoimmune T1D mouse models. This has prompted us to investigate whether altered functions of DC subsets contribute to the break of immune tolerance in autoimmune mouse models. In this study, characteristics of steady state DC subsets in the lymphoid organs of NOD mice were determined and compared with that of the diabetes resistant control mice NOR, Idd11 congenic mice (C57BL/6 x NOD intercross mice which have similar genetic background as NOD mice, but have a difference diabetes incidence level), as well as C57BL/6 mice. In addition, the frequency and the number of nTregs in NOD mice were also analyzed. A reduced percentage of splenic CD8+ cDC and an increased number of pDC in the thymus and spleen were found in NOD mice. Data showed that a CD8+ cDC deficiency, which is associated with diabetes onset in NOD mice, maps to the Idd11 locus. Further analysis of alterations in DC function and their potential contribution to the impairment of immune tolerance in NOD mice was carried out. A novel auto-antigen cross-presentation assay using islet-specific glucose-6-phosphate catalytic subunit-related protein (IGRP) was established to test the cross-presentation capacity of NOD DC. The results demonstrated a comparable cross-presentation ability between NOD and NOR cDC on a per cell basis. However, a lower capacity to cross-present cell associated auto-antigen in the spleen was found in NOD mice. Moreover, an elegant protocol, in which cells with the ability to cross-present can be deleted by horse cytochrome c (cyt c) treatment, was employed for the detection of cross-presenting cells. Results showed that the cells in NOD mice were insensitive to cyt c treatment, implying that the reduced percentage of the CD8+ cDC in NOD spleen was most likely due to the absence of the cDC subset with cross-presentation potential. These observations suggested that the reduced number of cross-presenting CD8+ cDC in NOD mice might result in a deficiency in maintaining peripheral immune tolerance with an accumulation of auto-reactive T cells in the peripheral, which could lead to the development of diabetes in NOD mice. pDC is a subset of DC that can protect the host from viral infection through the production of large amounts of type-1 interferon, including IFN-α, IFN-β etc., to inhibit the replication of viruses. The results from this study showed that pDC in NOD mice produced higher levels of IFN-α and IL-3 upon TLR ligand stimulation. Subsequent analysis showed that high dose of IFN-α could inhibit the proliferation of pathogenic CD8+ T cells. In addition, IL-3 could promote the development of CD103+ cDC with the ability to cross-present auto-antigens for the deletion of auto-reactive T cells in the peripheral tissues. These observations may help to explain the findings, which showed that infection by microbial agents could delay or prevent the onset of diabetes in NOD mice. Moreover, this study also revealed a reduction in the number of T regulatory cells in pancreatic lymph nodes, but not in the thymus and spleen of NOD mice. However, the ratio of Foxp3+ Tregs to CD4+ T cells was lower in thymus, spleen as well as in pancreatic lymph nodes of NOD mice. These findings suggests that the reduced ratio of Tregs to CD4+ T cells, rather than reducing absolute number of Tregs may result in less efficient control of auto-reactive T cells, and these may contribute to the development of diabetes in NOD mice. Overall, this study thoroughly examined and compared the phenotype, the number and the function of individual resident DC subsets in the thymus and spleen of NOD, NOR and Idd11 congenic mice. This study provides novel evidence for the importance of cross-presenting CD8+ cDC in the maintenance of peripheral immune tolerance. In addition, the distinct capacities of NOD pDC to produce IL-3 and much higher level of IFN-α in response to TLR activation were first demonstrated in this study. The effects of these two cytokines on T cells and DC function further suggested a potential mechanism for pDC in the prevention of T1D upon microbial stimulation. Further investigation of DC biology in T1D patients will be crucial for developing an effective approach for T1D immunotherapy

Study on the Role of ATM in T Cell Activation

ATM (Ataxia-Telangiectasia Mutated) 基因的突變與造成人類隱性遺傳疾病 A-T (Ataxia-Telangiectasia) 有關。ATM會參與 DNA破壞時的感應。A-T病患和 ATM缺失的老鼠會出現神經性疾病、胸腺細胞發育不全、較高的癌症罹患率和離子輻射敏感等現象。近期報告顯示ATM可抑制造血幹細胞的氧化壓力，維持其自我新生的能力。ATM還參與 T細胞發育階段 V(D)J基因重組時 DNA斷裂處的有效接合反應。但在成熟 T細胞中，ATM所扮演的角色目前並不清楚。報告顯示 T細胞在受到 PHA、EGF或 superantigen等物質的刺激時，其蛋白質含量及基因的表現上會有不同程度的變化。表示 ATM也可能在 T細胞的活化反應上扮演著某些調控功能。為了解 ATM在 T細胞活化過程中所可能扮演的角色，我們利用慢病毒 shRNA系統下調 Jurkat T細胞內生性 ATM，以探討此問題。 下調 Jurkat T細胞內生性 ATM，會顯著增加T細胞活化後所產生的 IL-2。進一步分析已知的幾條 T細胞活化訊息傳遞路徑，我們發現細胞活化早期 PKC-theta 的磷酸化會有部分增加。但其下游 NFkB活化後轉位到細胞核的程度並不明顯。對LAT、Akt、ERK及鈣離子相關訊息等都沒有顯著的影響。在 T細胞活化時，ATM蛋白質量與 Ser1981的磷酸化也都沒改變。歸結以上實驗得知，在 T細胞短暫活化過程中 PKC-theta 的磷酸化會部份受到ATM下調的影響，但其詳細的分子調控機制尚不清楚。此研究有助於我們更進一步了解除了 DNA斷裂的反應外，ATM也在 T細胞活化上可能扮演抑制性的角色。但對於 ATM在 T細胞活化的調控機制，還有待進一步的研究加以釐清。The Ataxia-Telangiectasia Mutated (ATM) protein kinase, mutations of which are associated with the human disease ataxia-telangiectasia (A-T), mediates responses to DNA damage in mammalian cells. A-T patients and Atm-deficient mice display neurologic dysfunction, defects in T lymphocyte maturation, cancer predisposition and radiation sensitivity. Recent study showed ATM protects bone marrow hemotapoietic stem cells (HSC) from oxidative stress and is required for sustained adult lymphopoiesis. Moreover, ATM routine surveillance of intermediates in V(D)J recombination, which is essential for TCR rearrangement. However, the role of ATM in mature T cells function is still unclear recently. Some reports showed that stimulation of T cells by using PHA, EGF or superantigen is able to alter ATM protein or gene expression level to some extent. Therefore, a general hypothesis emerges that ATM is involved in regulation of T cell activation. To test this hypothesis, we used lentiviral shRNA system to knockdown endogenous ATM in Jurkat T cells. Downregulation of ATM in Jurkat T cell cause dramatically increase in IL-2 production after stimulated with either OKT-3 plus anti-CD28 or SEE superantigen. To further study the molecular mechanisms, we found a partial effect on PKC-theta activation at early time point. However, the nuclear translocation of NFkB is not affected. LAT, Akt, ERK and calcium-related signals show no significant difference. Moreover, there is no change either in ATM protein expression level or ATM Ser1981 phosphorylation during T cell activation. In summary, knockdown ATM partially activate PKC-theta upon short-term stimulation, but the detail mechanisms are still unclear. This study provides information other than respondses to DNA damage, ATM may play a suppresive role in T cell activation, but the regulatory mechanism need to be clarified extensively.中文摘要............................................ i 英文摘要............................................ ii 目錄................................................ iv 第一章 簡介........................................ 1 1.1 Ataxia-Telangiectasia與Ataxia-Telangiectasia Mutated 簡介................................................. 1 1.1.1 Ataxia-Telangiectasia (A-T)........... 1 1.1.2 Ataxia-Telangiectasia Mutated (ATM)... 1 1.2 T細胞的發育 (T cell development)................. 4 1.3 ATM與T細胞的關係................................. 6 1.4 T細胞的活........................................ 8 1.5 研究方向與目的.................................. 10 第二章 材料與方法................................. 11 2.1 細胞株與培養.................................... 11 2.2 藥品配製與試劑.................................. 11 2.3 抗體............................................ 12 2.4小分子干擾核醣核酸下調 (shRNA knockdown)......... 12 2.4.1小分子干擾核醣核酸設計與質體構築...... 13 2.4.2 Lentivirus反轉錄病毒製備............. 14 2.4.3 Lentivirus反轉錄病毒效價測定......... 15 2.4.4 Lentivirus反轉錄病毒感染............. 16 2.5T細胞的活化...................................... 16 2.5.1固定式 anti-CD3/anti-CD28活化刺激..... 16 2.5.2SEE (Staphylococcal Enterotoxin E) 活化刺 激.................................... 17 2.6IL-2產量分析 (IL-2 assay)........................ 17 2.7流式細胞儀分析 (Flow cytometry analysis)細胞表面染 色............................................... 18 2.8細胞萃取液的製備................................. 18 2.8.1全細胞萃取液的製備.................... 18 2.8.2細胞核萃取液的製備.................... 19 2.9西方點墨法 (Western Blot)........................ 19 2.10鈣離子訊號的偵測 (Measurement of Ca2+ influx) .. 20 第三章 結果....................................... 22 3.1 ATM shRNA 成功下調JE6.1細胞內生性 ATM........... 22 3.2下調ATM使 T細胞活化 IL-2分泌量增加............... 23 3.3下調ATM對 T細胞活化訊息傳遞分子的影響............. 25 3.3.1 ATM下調不影響 LAT的活化.............. 25 3.3.2 ATM下調影響 PKC-theta活化早期的磷酸化 26 3.3.3 ATM下調不影響 NFκB細胞核轉位......... 26 3.3.4 ATM下調對 Akt活化的影響不明顯........ 27 3.3.5 ATM下調對 JNK、ERK MAPK活化的影響... 28 3.3.6 ATM下調對鈣離子訊息及下游分子 NFATc活化之分 析................................... 29 3.4 T細胞活化不影響 ATM表現......................... 30 3.5 ATM Ser1981磷酸化不參與T細胞的活化.............. 31 3.6 ATM的下調不影響T細胞表面 CD3分子的表現.......... 32 第四章 討論....................................... 33 圖表................................................ 38 參考文獻............................................ 59 附錄................................................ 6

Manipulation checks in hypertext experimental studies

Many experimental studies in hypertext exclude the use of manipulation checks. This study attempts to assess the effectiveness of including manipulation checks in hypertext experimental research

Inhibition of c-Rel expression in myeloid and lymphoid cells with distearoyl -phosphatidylserine (DSPS) liposomal nanoparticles encapsulating therapeutic siRNA.

c-Rel, a member of the nuclear factor kappa B (NF-κB) family, is preferentially expressed by immune cells and is known to regulate inflammation, autoimmune diseases and cancer. However, there is a lack of therapeutic intervention to specifically inhibit c-Rel in immune cells. Recent success with Pfizer and Moderna mRNA lipid-encapsulated vaccines as well as FDA approved medicines based on siRNA prompted us to test a lipid nanoparticle-based strategy to silence c-Rel in immune cells. Specifically, we encapsulated c-Rel-targeting siRNA into distearoyl-phosphatidylserine (DSPS)-containing nanoparticles. DSPS is a saturated phospholipid that serves as the "eat-me" signal for professional phagocytes such as macrophages and neutrophils of the immune system. We demonstrated here that incorporation of DSPS in liposome nanoparticles (LNP) improved their uptake by immune cells. LNP containing high concentrations of DSPS were highly effective to transfect not only macrophages and neutrophils, but also lymphocytes, with limited toxicity to cells. However, LNP containing low concentrations of DSPS were more effective to transfect myeloid cells than lymphoid cells. Importantly, DSPS-LNP loaded with a c-Rel siRNA were highly effective to inhibit c-Rel expression in several professional phagocytes tested, which lasted for several days. Taken together, our results suggest that DSPS-LNP armed with c-Rel siRNA could be exploited to target immune cells to limit the development of inflammatory diseases or cancer caused by c-Rel upregulation. In addition, this newly developed DSPS-LNP system may be further tested to encapsulate and deliver other small molecule drugs to immune cells, especially macrophages, neutrophils, and lymphocytes for the treatment of diseases

Design of Feedback Control for Networked Finite-Distributed Delays Systems with Quantization and Packet Dropout Compensation

This paper investigates the feedback control for networked discrete-time finite-distributed delays with quantization and packet dropout, and systems induce the H∞ control problem. The compensation scheme occurs in a random way. The quantization of system state or output signal is in front of being communicated. It is shown that the design of both a state feedback controller and an observer-based output feedback controller can be achieved, which ensure the asymptotical stability as well as a prescribed H∞ performance of the resulting closed-loop system satisfying dependence on the size of the discrete and distributed delays. Numerical examples are given to illustrate the effectiveness and applicability of the design method in this paper